The vast numbers of transposable elements littering our genomes are thought to have contributed to genome evolution on the one hand and genome instability on the other. The long term goal of this research is to understand the mechanisms underlying the genetic success of these elements. Phage Mu has a served as a paradigm for transposition for a quarter of a century. While much has been learned about the chemistry of phosphoryl transfer, much remains to be learned about the mechanism and regulation of transposition including transpososome assembly, control of directionality, and target DNA selection. Almost nothing is known about the integration mechanism of the linear Mu virion DNA into the host chromosome, a process with many similarities to linear HIV DNA integration. We plan to build on the major advances in our last grant proposal, as well as our preliminary new results, to propose the following three specific aims: (1) Dissect the detailed architecture of the 5-noded 3-site Mu synaptic complex, and understand the role of the enhancer in its assembly. (2) Design experiments to understand the opposing activities of target site selection and target immunity. (3) Lay the groundwork for analyzing how flanking host sequences are processed upon integration of infecting Mu DNA in vivo, which occurs by a variation of the established cointegrate mechanism. These studies will provide important new perspectives on several fundamental aspects of the movement of transposable elements, and will contribute not only to understanding their insistent presence throughout the biological world, but also to how they affect human health. Mobile genetic elements impact our daily lives in significant ways. On the one hand, DNA transposition is responsible for the transmission of drug resistance in bacteria, integration of retroviral genomes into host chromosomes, and genome instabilities leading to cancer. On the other hand, transposition mechanisms have been widely exploited as tools for genetic engineering and in strategies for gene therapy. Our studies are expected to impact both areas of research.